Use of cysteine derivatives for the preparation of a medicament intended to treat pathologies which result from the formation of the heterotrimeric G protein

ABSTRACT

The invention relates to the use of cysteine derivatives for preparing a medicament intended to treat diseases which result from the formation of the heterotrimeric G protein. These diseases include in particular diseases linked to the following biological functions or disorders: smell, taste, perception of the light, neurotransmission, neurodegeneration, endocrine and exocrine gland functions, autocrine and paracrine regulation, arterial tension, embryogenesis, benign cell proliferation, oncogenesis, viral infection, immunological functions, diabetes, obesity, and benign and malign proliferative diseases.  
     Said cysteine derivatives include in particular:  
     bis-1,1′-[7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine disulphide (I), and  
     bis-1,1′-7-(2-amino-1-oxo-3-thiopropyl-(2-(1-naphtyl)-8-(2-methylpropyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazin-7-yl) disulphide (II).

[0001] The present invention relates in particular to the use ofderivatives of cysteine for the preparation of a medicament intended totreat pathologies which result from the formation of the heterotrimericG protein. These diseases include in particular diseases linked to thefollowing biological functions or disorders: smell, taste, perception oflight, neurotransmission, neurodegeneration, endocrine and exocrinegland functions, autocrine and paracrine regulation, arterial tension,embryogenesis, benign cell proliferation, oncogenesis, viral infection,immunological functions, diabetes, obesity, and benign and malignproliferative diseases.

[0002] The G proteins are in fact the structural association of threedistinct sub-units called a, β and γ, but operate as dissociableentities constituted by α sub-units on the one hand and β/γ dimers onthe other hand.

[0003] The G proteins participate in the transmission of signals outsidethe cell thanks to its interaction with receptors with seventransmembrane domains inside using different effectors includingadenylate cyclase, phospholipase C or also the ionic channels. Theadenylate cyclase enzyme generates cyclic AMP (cAMP) (cf. Gilman, A. G.Biosci. Rep. 15, 65-97 (1995)). Thus, it is known that, in order toactivate the adenylate cyclase, it is necessary for the G proteins to betransitionally in a heterotrimeric form, in which form the monomerconstituted by an α sub-unit is associated with the dimer constituted bythe β and γ sub-units. It is only in this situation that the signaloutside the cell can activate the α sub-unit of a G protein, which can,after disassociation, modulate the adenylate cyclase and modulate theproduction of cAMP.

[0004] It is also known that the β/γ dimers can directly activate theeffectors leading to the activation of kinases regulated byextracellular signals (ERKs) or MAP kinases. A direct link between theβ/γ sub-units and the src or src like kinases has been demonstrated (cf.Gutkind, J. S. J. Biol. Chem. 273, 1839-1842 (1998)).

[0005] Moreover, bacterial toxins such as Vibrio cholera and Bortellapertussis, peptides such as mastoparan and suramin have been presentedas directly modulating the activity of the G proteins (cf. Freissmuth,M., Boehm, S., Beindl, W., et al. Mol. Pharmacol. 49, 602-611 (1996);Boehm, S., Huck, S., Motejlek, A., et al. Journal of Neurochemistry 66,1019-1026 (1996); Cachero, T. G., Rigual, R., Rocher, A. & Gonzalez, C.Eur. J. Neurosci. 8, 2320-2327 (1996); Danilenko, M., Worland, P.,Carlson, B., Sausville, E. A. & Sharoni, Y. Biochem. Biophys. Res.Commun. 196, 1296-1302 (1993); Beindl, W., Mitterauer, T., Hohenegger,M., Ijzerman, A. P., Nanoff, C. & Freissmuth, M. Mol. Pharmacol. 50,415-423 (1996)).

[0006] For example, the choleric toxin modifies the α_(S) sub-unit ofthe G protein by adding an ADP-ribose originating from the NAD to anarginine-specific acceptor site. This completely blocks the activity ofthe GTPase, provoking persistent stimulation of its next effector,adenylate cyclase and leading to overproduction of cAMP.

[0007] The harmful effects of an abnormal cAMP level are also known andoccur in particular at the level of the following biological functionsor disorders: smell, taste, perception of light, neurotransmission,neurodegeneration, endocrine and exocrine gland functions, autocrine andparacrine regulation, arterial tension, embryogenesis, benign cellproliferation, oncogenesis, viral infection and immunological functions,diabetes and obesity.

[0008] The Applicant has just discovered that certain derivatives ofcysteine, namely the compounds of general formula (A)

[0009] corresponding to sub-formulae (A1) or (A2):

[0010] in which:

[0011] X represents R₁₂ and Y represents R₈, or X and Y complete a ringwith 6 members, the X—Y set representing the —CH(R₈)—CH(R₉)— radical;

[0012] R₁ represents H, a lower alkyl or alkylthio radical;

[0013] R₂ and R₃ represent independently H or a lower alkyl radical;

[0014] R₄ represents H₂ or O;

[0015] R₅ represents H, or one of the lower alkyl, lower alkenyl, loweralkynyl, aryl, lower arylalkyl, heterocycle or lower alkyl heterocycleradicals, these radicals being optionally substituted by radicals chosenfrom the group comprising a lower alkyl radical, —O—R₁₀, —S(O)_(m)R₁₀ (mrepresenting 0, 1, or 2), —N(R₁₀)(R₁₁), —N—C(O)—R₁₀, —NH—(SO₂)—R₁₀,—CO₂—R₁₀, C(O)—N(R₁₀)(R₁₁), and —(SO₂)—N(R₁₀)(R₁₁);

[0016] R₆ and R₇ represent independently H, a —C(O)—NH—CHR₁₃—CO₂R₁₄radical, or one of the lower alkyl, aryl, lower arylalkyl, heterocycleor lower alkyl heterocycle radicals, these radicals being optionallysubstituted by radicals chosen from the group comprising the OH, alkylor lower alkoxy, N(R₁₀)(R₁₁), COOH, CON(R₁₀)(R₁₁), and halo radicals,

[0017] or R₆ and R₇ form together an aryl radical or a heterocycle;

[0018] R₈ and R₉ represent independently, H, or one of the lower alkyl,aryl, lower arylalkyl, heterocycle or lower alkyl heterocycle radicals,these radicals being optionally substituted by radicals chosen from thegroup comprising the OH, alkyl or lower alkoxy, N(R₁₀)(R₁₁), COOH,CON(R₁₀)(R₁₁) and halo radicals, or R₈ and R₉ together form an arylradical or a heterocycle;

[0019] R₁₀ and R₁₁ represent independently H, an aryl radical or aheterocycle, or an alkyl, arylalkyl or lower alkyl heterocycle radical;

[0020] R₁₂ represents NR₉, S, or O;

[0021] R₁₃ represents a lower alkyl radical optionally substituted by aradical chosen from the lower alkyl, —OR₁₀, —S(O)_(m)R₁₀ (m representing0, 1, or 2) and —N(R₁₀)(R₁₁) radicals;

[0022] R₁₄ represents H or a lower alkyl radical;

[0023] or the compounds of general formula (B);

W₁—Ar—W₂  (B)

[0024] in which:

[0025] W₁ represents a remainder originating from a cysteine in reducedor non reduced form;

[0026] Ar represents a radical derived from an aminobenzoic acid, thearomatic ring of which is optionally substituted;

[0027] W₂ represents an amino acid, preferably an aliphatic amino acid;

[0028] or also the compounds of general formula (C):

[0029] in which:

[0030] Z₁ represents a lower alkyl radical;

[0031] Z₂ and Z₃ both represent H or Z₂ and Z₃ together form a chainhaving 2 to 4 elements chosen from the —C(O)—, —CH₂—, —CH(NH₂)— and —S—radicals, it being understood that two successive elements are not both—C(O)—;

[0032] it being understood that the compounds of general formula (C) canalso be presented in the form of dimers, when the Z₂ radical representsa hydrogen atom which can be eliminated by oxidization;

[0033] or also a pharmaceutically acceptable salt of a compound ofgeneral formula (A), (B) or (C);

[0034] can be used to prepare medicaments intended to treat pathologieswhich result from the formation of the heterotrimeric G protein.

[0035] By lower alkyl radical, is understood a linear or branched alkylradical containing 1 to 6 carbon atoms, and in particular the methyl,ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and tert-butyl,pentyl, neopentyl, isopentyl, hexyl, isohexyl radicals. By heterocycleradical is understood a radical constituted by one or more rings andincluding at least one heteroatom. By arylalkyl, alkyl heterocycle,alkylthio or lower alkoxy radical, is understood the radicals of whichthe alkyl radical has the meaning indicated previously.

[0036] Preferably, the Ar radical included in formula (B) is optionallysubstituted by an alkyl radical comprising 1 to 6 carbon atoms or anaryl radical, these alkyl or aryl radicals themselves being optionallysubstituted preferentially by an alkoxy radical having 1 to 4 carbonatoms, fluoro, chloro, bromo. The aryl radical preferably a phenyl canitself be substituted by an alkyl radical.

[0037] Preferably also, the compounds of general formula (B) are suchthat Ar represents a radical derived from an aminobenzoic acid thearomatic ring of which is substituted by a phenyl radical and W₂represents an aliphatic amino acid.

[0038] In particular, the following compounds can be used to preparemedicaments intended to treat pathologies which result from theformation of the heterotrimeric G protein:

[0039]7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methylphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;

[0040]7-(2-amino-1-oxo-3-thiopropyl)-8-butyl-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;

[0041]7-(2-amino-1-oxo-3-thiopropyl)-8-(1-methylpropyl)-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;

[0042]1-[2(R)-amino-3-mercaptopropyl]-2(S)-n-butyl-4-(1-naphthoyl)piperazine;

[0043]bis-1,1′-[7-(2-amino-1-oxo-3-thiopropyl)-2-(methoxyphenyl)-8-(1-methylpropyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine]disulphide;

[0044]bis-1,1′-[7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazinedisulphide;

[0045]bis-1,1′-7-(2-amino-1-oxo-3-thiopropyl-(2-(1-naphthyl)-8-(2-methylpropyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazin-7-yl)disulphide;

[0046] the compound of formula:

[0047] the compound of formula:

[0048]7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-phenyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;

[0049]7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenylmethoxy)methyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;

[0050]7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(1-phenylmethoxy)ethyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;

[0051]7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenoxyethyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;

[0052]7-(2-amino-l-oxo-3-thiopropyl)-2-(2-methyoxyphenyl)-8-(phenoxyethyl)-5,6,7,8-tetrahydro-imidazo[1.2a]pyrazine,or its dimeric form;

[0053] and7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenylsulphonylethyl)-5,6,7,8-tetrahydro-imidazo[1.2a]pyrazine;

[0054] or also a pharmaceutically acceptable salt of one of thesecompounds.

[0055] One of the following compounds is preferably used for theinvention:

[0056]bis-1,1′-[7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazinedisulphide (I);

[0057]bis-1,1′-7-(2-amino-1-oxo-3-thiopropyl-(2-(1-naphthyl)-8-(2-methylpropyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazin-7-yl)disulphide (II);

[0058]7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methylphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine(III);

[0059] the compound of formula:

[0060]7-(2-amino-1-oxo-3-thiopropyl)-8-butyl-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine(V);

[0061]bis-1,1′-[7-(2-amino-1-oxo-3-thiopropyl)-2-(methoxyphenyl)-8-(1-methylpropyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine]disulphide (VI),

[0062] the compound of formula:

[0063]7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-phenyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;

[0064]7-(2-amino-1-oxo-3-thiopropyl)-8-(1-methylpropyl)-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;

[0065]1-[2(R)-amino-3-mercaptopropyl]-2(S)-n-butyl-4-(1-naphthoyl)piprazine.

[0066] or a pharmaceutically acceptable salt of one of the latter.

[0067] More preferentially, one of the following compounds is used forthe invention:

[0068]bis-1,1′-[7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazinedisulphide (I);

[0069]bis-1,1′-7-(2-amino-1-oxo-3-thiopropyl-(2-(1-naphthyl)-8-(2-methylpropyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazin-7-yl)disulphide (II);

[0070]7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methylphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine(III);

[0071]7-(2-amino-1-oxo-3-thiopropyl)-8-butyl-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine(V);

[0072] the compound of formula:

[0073] or a pharmaceutically acceptable salt of one of the latter.

[0074] Finally, the following compounds are more particularly preferred:

[0075]bis-1,1′-[7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazinedisulphide (I);

[0076]bis-1,1′-7-(2-amino-1-oxo-3-thiopropyl-(2-(1-naphthyl)-8-(2-methylpropyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazin-7-yl)disulphide (II);

[0077] or a pharmaceutically acceptable salt of one of the latter.

[0078] The invention therefore relates firstly to the use of thecompounds of general formula (A), (B) or (C) as described previously forpreparing a medicament intended to treat pathologies which result fromthe formation of the heterotrimeric G protein. In particular, it relatesto the use of said inhibitors for preparing medicaments intended totreat diseases linked to the following biological functions ordisorders: smell, taste, perception of light, neurotransmission,neurodegeneration, endocrine and exocrine gland functions, autocrine andparacrine regulation, arterial tension, embryogenesis, viral infection,immunological functions, diabetes and obesity.

[0079] More particularly, the invention relates to the use of compoundsof general formula (A), (B) or (C) for preparing a medicament intendedto treat cholera, Acquired Immune Deficiency Syndrome (AIDS), traveldiarrhea and familial masculine precocious puberty.

[0080] A subject of the invention is also new products of generalformula (A) numbered 1 to 7 and described hereafter in the examples,namely:

[0081]7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methylphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;

[0082]7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-phenyl-5,6,7,8-tetrahydroimidazo[10.2a]pyrazine;

[0083]7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenylmethoxy)methyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;

[0084]7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(1-phenylmethoxy)ethyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;

[0085]7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenoxyethyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;

[0086] 7-(2-amino1-3-thiopropyl)-2-(2-methyoxyphenyl)-8-(phenoxyethyl)-5,6,7,8-tetrahydro-imidazo[1.2a]pyrazine,or its dimeric form;

[0087] and7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenylsulphonylethyl)-5,6,7,8-tetrahydro-imidazo[1.2a]pyrazine.

[0088] A subject of the invention is also said new products or theirpharmaceutically acceptable salts as medicaments, as well as their usefor preparing a medicament intended to treat pathologies which resultfrom the formation of the heterotrimeric G protein. In particular, itrelates to the use of said products for preparing medicaments intendedto treat diseases linked to the following biological functions ordisorders: smell, taste, perception of light, neurotransmission,neurodegeneration, endocrine and exocrine gland functions, autocrine andparacrine regulation, arterial tension, embryogenesis, benign cellproliferation, oncogenesis, viral infection, immunological functions,diabetes, obesity, and benign and malign proliferative diseases.

[0089] The products particularly preferred for use according to theinvention are therefore the following:

[0090]bis-1,1′-[7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazinedisulphide;

[0091]bis-1,1′-7-(2-amino-1-oxo-3-thiopropyl-(2-(1-naphthyl)-8-(2-methylpropyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazin-7-yl)disulphide;

[0092] the compound of formula:

[0093]7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methylphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;

[0094]7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-phenyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;

[0095]7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenylmethoxy)methyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;

[0096]7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(1-phenylmethoxy)ethyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;

[0097]7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenoxyethyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;

[0098]7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methyoxyphenyl)-8-(phenoxyethyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine,or its dimeric form;

[0099] and7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenylsulphonylethyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;

[0100] or a pharmaceutically acceptable salt of one of the latter.

[0101] Similarly, the invention relates more particularly to the use ofthe compounds previously mentioned for preparing a medicament intendedto treat cholera, Acquired Immune Deficiency Syndrome (AIDS), traveldiarrhea and familial masculine precocious puberty.

[0102] The compounds of general formula (A) and their preparation aredescribed in the Patent Application WO 97/30053 or in the exampleshereafter. The compounds of general formula (B) and their preparationare described in the Patent Application WO 96/21456. Finally, thepreparation of the compounds of general formula (C) is described in thePatent Application PCT WO 95/00497, except for the compound of formula(VII) for which the synthesis is described in the experimental part ofthis Application.

[0103] The pharmaceutical compositions comprising a compound of theinvention can be in the form of solids, for example powders, granules,tablets, gelatin capsules, liposomes or suppositories. The appropriatesolid supports can be, for example, calcium phosphate, magnesiumstearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose,methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidineand wax.

[0104] The pharmaceutical compositions comprising a compound of theinvention can also be presented in liquid form, for example, solutions,emulsions, suspensions or syrups. The appropriate liquid supports canbe, for example, water, organic solvents such as glycerol or glycols, aswell as their mixtures, in varying proportions, in water.

[0105] The administration of a medicament according to the invention canbe carried out by topical, oral, parenteral route, by injection(intramuscular, sub-cutaneous, intravenous, etc.), etc. Theadministration route will of course depend on the type of disease to betreated.

[0106] The administration dose envisaged for a medicament according tothe invention is comprised between 0.1 mg and 10 g depending on the typeof pathology to be treated.

[0107] Unless they are defined in another manner, all the technical andscientific terms used here have the same meaning as that usuallyunderstood by an ordinary specialist in the field to which thisinvention belongs. Similarly, all the publications, patent applications,all the patents and all other references mentioned here are incorporatedby way of reference.

EXAMPLES Example 17-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methylphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine:1

[0108] Compound 1 was prepared according to the synthesis diagram below:

[0109] 1.a) Carbobenzyloxy-L-cyclohexylalanine

[0110] L-phenylalanine (10.0 g; 60.6 mmol) is combined with PtO2 (430mg) in acetic acid (60 ml) and the mixture is hydrogenated overnightunder 20-50 psi H₂. A 5% aqueous solution of HCl is added to the mixturein order to obtain a limpid solution and hydrogenation is continueduntil the consumption of hydrogen ceases. The catalyst is eliminated byfiltration and the filtrate is concentrated under reduced pressure. Theresidue is taken up in methanol and water and the pH is adjusted to 4.4by the addition of a 10% solution of NaOH. The product obtained isrecovered by filtration and used without further purification.

[0111] L-cyclohexylalanine (60.6 mmol) is suspended in water (100 ml),K₂CO₃ (8.36 g; 60.6 mmol) is added, then a solution ofN-(benzyloxycarbonyloxy)succinimide (15.1 g; 60.6 mmol) in CH₃CN (150ml) and the mixture obtained is agitated vigorously for 45 minutes. Themixture is concentrated in order to produce a volume of approximately100 ml and washed with Et₂O (100 ml), then acidified with concentratedHCl and extracted with AcOEt (2×50 ml). The combined AcOEt phases aredried over Na₂SO₄, filtered and concentrated in order to produce a clearoil (17.27 g; 93%).

[0112] NMR ¹H (DMSO-d6): 7.5-7.6 (1H, d); 7.2-7.5 (5H, m); 5.0-5.1 (2H,s); 3.9-4.1 (1H, m); 0.7-1.8 (13H, m).

[0113] 1.b)2-(]-(S)-((phenylmethoxy)carbonyl)-amino-2-(cyclohexyl)methyl)-4-(2-methylphenyl)-imidazole

[0114] Cbz-(L)-cyclohexylalanine (4.58 g; 15.0 mmol) and Cs₂CO₃ (2.44 g;7.50 mmol) are placed in a 2:1 mixture of DMF:H₂O (75 ml). The mixtureobtained is agitated until it becomes homogeneous. The solvents areeliminated under reduced pressure, the residue is dissolved in DMF (60ml) and 2-bromo-2′-methylacetophenone (3.20 g; 15.0 mmol) in DMF (30 ml)is added. The mixture is agitated overnight at ambient temperature thenfiltered and concentrated under reduced pressure. The keto-esterobtained is solubilized in xylenes (100 ml) and ammonium acetate (19.5g; 0.25 mol) is added. The mixture is heated to reflux for approximately3 hours with elimination of the excess AcONH₄ supernatant and of thewater released by means of a Dean-Stark trap. The reaction mixture isconcentrated under reduced pressure, taken up in AcOEt and washed with asaturated solution of NaHCO₃ (100 ml) and with a saturated solution ofNaCl (100 ml). The AcOEt phase is dried over Na₂SO₄, filtered andconcentrated under vacuum. The crude product obtained is purified byflash chromatography on silica gel with a CHCl₃/MeOH mixture 98/2 aseluant. The fractions containing the pure product are combined andconcentrated in order to produce the product (2.52 g; 40%) in the formof a slightly brown foam which is used in the following stage withoutadditional purification.

[0115] 1.c)2-(1-(S)-((phenylmethoxy)carbonyl)-amino-2-(cyclohexyl)methyl)-1-((2-ethoxy-2-oxo)ethyl)-4-(2-methylphenyl)-imidazole

[0116] Intermediate 1.b (2.52 g; 6.0 mmol) is solubilized in DMF (20 ml)and treated with K₂CO₃ (1.67 g, 12.1 mmol) and ethyl bromoacetate (1.34ml; 12.5 mmol) is added. The mixture obtained is heated at 45° C. forone and a half hours. The mixture is diluted with ether (50 ml) andwashed with a saturated solution of NaHCO₃ solution (50 ml) then with asaturated solution of NaCl (50 ml). The ethereal layer is dried overNa₂SO₄, filtered and concentrated in order to produce an oil which isused in the following stage without additional purification.

[0117] Mass spectrum: 504.3 MH+.

[0118] 1.d)8-(cyclohexylmethyl)-6-oxo-2-(2-methylphenyl)-imidazo[1,2-a]pyrazine

[0119] The crude intermediate of Stage 1.c is solubilized in acetic acid(50 ml) containing a 10% Pd on carbon catalyst (152 mg), thenhydrogenated under a pressure of 50 psi of H₂ for 18 hours at ambienttemperature. The catalyst is eliminated by filtration and the filtrateis heated at 70° C. for 2 hours. The mixture obtained is concentratedunder reduced pressure, dissolved in CH₂Cl₂ (100 ml) and washed with asaturated solution of NaHCO₃ (100 ml). The CH₂Cl₂ layer is dried overNa₂SO₄, filtered and concentrated in order to produce a viscous oilwhich is used in the following stage without additional purification.

[0120] Mass spectrum: 324.3 MH+.

[0121] 1.e)8-(cyclohexylmethyl)-2-(2-methylphenyl)-4,5,6,7-teirahydro-imidazo[1,2-a]pyrazine

[0122] The crude intermediate of Stage 1.c is solubilized in THF (25 ml)and treated at ambient temperature with a 1 M solution of BH3 in THF (25ml) for half an hour then taken to reflux for 1 hour. The mixture iscooled down using an ice bath and 4N HCl (40 ml) is added dropwise at 0°C. The mixture is taken to ambient temperature then taken to reflux for1 hour. The reaction medium is then cooled down, filtered andconcentrated under reduced pressure. The residue is treated with asaturated solution of NaHCO₃ (50 ml) and extracted with CH₂Cl₂ (3×50ml). The CH₂Cl₂ phases are dried over Na₂SO₄, filtered and concentratedin order to produce a slightly brown oil (1.63 g; yield of 87% relativeto Stages 1.c, 1.d and 1.e).

[0123] Mass spectrum: 310.3 MH+.

[0124] 1.f)8-(cyclohexylmethyl)-7-[2-(((1,1-dimethylethoxy)carbonyl)amino)-1-oxo-3-((triphenylmethyl)thio)propyl]-2-(2-methylphenyl)-4,5,6,7-tetrahydro-imidazo-[1.2a]-piperazine

[0125] Diisopropylcarbodiimide (908 μl; 5.80 mmol) and BocCys(trt)-OH(5.37 g; 11.6 mmol) are solubilized in CH₂Cl₂ (25 ml), the mixtureobtained being agitated for 45 minutes.8-(cyclohexylmethyl)-2-(2-methylphenyl)-4,5,6,7-tetrahydro-imidazo[1,2-a]pyrazine(1.63 g; 5.27 mmol) is then added. The reaction mixture is agitatedovernight at ambient temperature. The solvent is eliminated underreduced pressure and the product obtained is purified by flashchromatography on silica gel with a CH₂Cl₂/MeOH mixture 98/2 as eluant.The pure fractions are concentrated in order to produce a viscous oilwhich is which is used in the following stage without additionalpurification.

[0126] Mass spectrum: 755.6 MH+.

[0127] 1.g)7-(2-amino-1-oxo-3-(mercaptopropyl))-8-(cyclohexylmethyl)-2-(2-methylphenyl)-4,5,6,7-tetrahydro-imidazo-[1.2a]-piperazine:1:

[0128] The intermediate of Stage 1.f (3.54 g; 4.69 mmol) is solubilizedin trifluoroacetic acid (TFA, 80 ml) containing triisopropylsilane (1.92ml; 9.38 mmol) and the reaction mixture is agitated at ambienttemperature under nitrogen for one hour. The reaction mixture isfiltered and the filtrate is concentrated under reduced pressure. Theresidue is extracted by trituration with an aqueous solution of TFA at0.1% (6×65 ml) and filtered. The crude product is purified bypreparative HPLC on a C18 column using a gradient of 0 to 20% of CH₃CNin an aqueous solution of TFA at 0.1% for 30 minutes. The pure fractionsof the product are collected and lyophilized. The initial product islyophilized twice from a dilute solution of HCl in order to produce theproduct in the form of its hydrochloride (740 mg; 32%).

[0129] Mass spectrum: 413.2 MH+. NMR ¹H (DMSO-d6): 8.5-9.0 (3H, d,broad); 7.8-8.0 (1H, s); 7.5-7.7 (1H, d); 7.2-7.5 (3H, m); 5.8-6.1 (1H,m); 4.65-4.8 (1H, s); 4.5-4.7 (1H, d); 4.1-4.4 (2H, m); 3.8-4.0 (1H, m);3.2-3.7 (H₂O); 2.8-3.1 (2H, m); 2.35-2.5 (3H, s); 2.0-2.2 (1H, m);1.8-2.05 (2H, m); 1.25-1.4 (4H, broad s); 1.3-0.9 (6H, m).

Example 27-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-phenyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine:2:

[0130] Compound 2 is prepared according to diagram 1, Stages b to g,according to a method similar to that of Example 1, 2-bromoacetophenonereplacing 2-bromo-2′-methylacetophenone in Stage b.

[0131] Mass spectrum: 399.2 MH+. NMR ¹H (DMSO-d6): 8.5-8.9 (3H, broadd); 8.0-8.2 (1H, s); 7.8-8.0 (2H, d); 7.45-7.56 (2H, t); 7.35-7.5 (1H,t); 5.9-6.05 (1H, broad s); 4.65-4.8 (1H, s); 4.5-4.65 (1H, d); 4.1-4.35(2H, m); 3.8-4.0 (1H, m); 3.2-3.8 (H₂O); 3.25-3.4 (1H, t); 2.8-3.05 (2H,m); 2.05-2.2 (1H, d); 1.85-2.05 (2H, t); 1.55-1.75 (4H, broad s);1.15-1.3 (1H, broad s); 1.2-0.9 (5H, m).

Example 37-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenylmethoxy)methyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine:3:

[0132] Compound 3 is prepared according to diagram 1, Stages b to g,according to a method similar to that of Example 1, Boc-(L)-Ser(Bzl)-OHreplacing Cbz-(L)-cyclohexylalanine in Stage b and Stage d beingreplaced by a deprotection using TFA and iPr₃SiH according to a methodsimilar to reaction 1.g. The product is obtained in the form of a pairof diastereoisomers in a proportion of 2:3.

[0133] Mass spectrum: 453.2 MH+.

[0134] The retention times for the diastereoisomers are 6.58 and 7.07minutes respectively in the following HPLC system: Eluant 30-50%CH₃CN/0.1% TFA Duration of elution: 24 minutes Detection 254 nm ColumnVydac protein and C18 peptide

Example 47-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(1-phenylmethoxy)ethyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine:4:

[0135] Compound 4 is prepared according to diagram 1, Stages b to g,according to a method similar to that of Example 3, Boc-(L)-Thr(Bzl)-OHreplacing Boc-(L)-Ser(Bzl)-OH in Stage b.

[0136] Mass spectrum: 467.3 MH+. NMR ¹H (DMSO-d6): 8.5-8.9 (3H, d,broad); 8.0-8.1 (1H, s); 7.95-8.1 (2H, d); 7.4-7.5 (1H, t); 7.15-7.3(1H, d); 7.0-7.2 (3H, m); 6.9-7.05 (2H, m); 5.85-5.95 (1H, d); 4.75-4.85(1H, broad s); 4.65-4.8 (1H, broad s); 4.35-4.65 (3H, m); 4.1-4.25 (2H,q); 3.9-4.0 (3H, s); 2.8-3.1 (2H, m); 1.2-1.4 (3H, d).

Example 57-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenoxyethyl)-5,6,7,8-tetrahydroimidazo[1,2a]pyrazine:5:

[0137] Compound 5 was prepared according to the following synthesisdiagram:

[0138] 5.h)2-(1-(S)-((phenylmethoxy)carbonyl)-amino-2-(2-oxo-2-(phenylmethoxy)ethyl)-4-(2-methoxyphenyl)-imidazole

[0139] Cbz-(L)-Asp(Obzl)-OH (5.00 g; 14.0 mmol) and Cs₂CO₃ (2.28 g; 7.00mmol) are mixed in a 1:1 mixture of DMF:H₂O (75 ml). The mixtureobtained is agitated until it becomes homogenous. The solvents areeliminated under reduced pressure, the residue is dissolved in DMF (60ml) and 2-bromo-2′-methoxyacetophenone (3.21 g; 14.0 mmol) in DMF (30ml) is added. The mixture obtained is agitated for half an hour atambient temperature then filtered and concentrated under reducedpressure. The keto-ester obtained is triturated with a 1:1 mixture ofEt₂O:hexanes (2×40 ml) then suspended in xylenes (100 ml). Ammoniumacetate (17.5 g; 0.23 mol) is added and the mixture is heated at refluxfor approximately one hour and 30 minutes with elimination of the excessAcONH₄ and of the water released by means of a Dean-Stark trap. Thereaction medium is washed with a saturated solution of NaHCO₃ (50 ml),dried over Na₂SO₄, filtered and concentrated under vacuum in order toproduce 6.66 g (98%) of desired product.

[0140] Mass spectrum: 486.3 (MH+).

[0141] 5.i)2-(1-(S)-((phenylmethoxy)carbonyl)-amino-2-((2-phenylmethoxy-2-oxo)ethyl)-1-((2-ethoxy-2-oxo)ethyl)-4-(2-methoxyphenyl)-imidazole

[0142] Intermediate 5.i is prepared according to a method similar tothat of Stage 1.c.

[0143] Mass spectrum: 572.3 MH+.

[0144] 5.j)(6-oxo-2-(2-methoxyphenyl)-5,6,7,8-tetrahydro-imidazo[1,2-a]pyrazin-8-yl)-aceticAcid

[0145] Intermediate 5.j is prepared according to a method similar tothat of Stage 1.d.

[0146] Mass spectrum: 302.2 MH+. NMR ¹H (DMSO-d6): 8.35-8.5 (1H, d,broad); 8.0-8.1 (1H, dd); 7.45-7.55 (1H, s); 7.15-7.25 (1H, m); 7.0-7.1(1H, m); 6.9-7.0 (1H, m); 4.85-5.0 (1H, broad s); 4.55-4.75; (2H, q);3.85-3.95 (3H, s); 2.8-2.95 (2H, d).

[0147] 5.k) 8-hydroxyethyl-2-(2-methoxyphenyl)-5, 6,7,8-tetrahydro-imidazo[1,2-a]pyrazine

[0148] Intermediate 5.k is prepared according to a method similar tothat of Stage 1.e, except for the fact that a molar proportion of 6/9 ofBH₃ relative to the substrate is used.

[0149] Mass spectrum: 274.3 MH+.

[0150] 5.l)7-((1,1-dimethylethoxy)carbonyl)-8-hydroxyethyl-2-(2-methyoxyphenyl)-5,6,7,8-tetrahydro-imidazo[1,2-a]pyrazine

[0151] Intermediate 5.k (1.36 g; 5.0 mmol) is suspended in H₂O (5 ml)and a mixture of di-t-butyldicarbonate (1.20 g; 5.5 mmol) in p-dioxane(10 ml) is added. The reaction medium is agitated vigorously andmaintained at pH 8.0-8.4 by the dropwise addition of a 2.5N solution ofNaOH until the reaction finishes (monitoring of the reaction by TLC onsilica gel, eluant AcOEt:hexanes 3:2). The crude product is purified byflash chromatography on silica gel with an AcOEt:hexanes mixture 3:2 aseluant (Biotage system, pre-filled columns 4×15 cm). The fractionscontaining the product are combined and concentrated under vacuum inorder to produce a white foam (1.60 g; 86%).

[0152] Mass spectrum: 374.3 MH+. NMR ¹H (DMSO-d6): 7.95-8.05 (1H, d,d);7.45-7.55 (1H, s); 7.10-7.25 (1H, m); 7.0-7.1 (1H, m); 6.9-7.05 (1H, m);5.05-5.15 (1H, t); 4.25-4.35 (1H, t); 4.05-4.2 (1H, broad s); 4.0-4.1(1H m); 3.9-4.0 (3H, s); 3.9-4.0 (1H, m); 3.25-3.35 (2H, m); 1.9-2.1(2H, m); 1.15-1.25 (9H, s).

[0153] 5.m)7-((1,1-dimethylethoxy)carbonyl)-8-phenoxyethyl-2-(2-methoxyphenyl)-5,6,7,8-tetrahydro-imidazo[1,2-a]pyrazine

[0154] Intermediate 5.1 (746 mg; 2.00 mmol) is dissolved in THF (10 ml)containing triphenylphosphine (550 mg; 2.1 mmol) and phenol (198 mg; 2.1mmol). The mixture is cooled down to 0° C. under nitrogen anddiethylazodicarboxylate (330 μl; 2.1 mmol) is added dropwise over 10minutes. The reaction mixture is then agitated for 2 hours at ambienttemperature. The reaction medium is then cooled down again to 0° C. andtriphenylphosphine (275 mg; 1.05 mmol) and phenol (99 mg; 1.05 mmol) areadded. Then diethylazodicarboxylate (166 μl; 1.05 mmol) is addeddropwise over 10 minutes then the mixture is agitated again for 1 hourat ambient temperature. The solvents are eliminated under reducedpressure and the crude product is purified by flash chromatography onsilica gel with an AcOEt:hexanes mixture 3:2 as eluant. The fractionscontaining the product are combined and concentrated under vacuum. Afterrecrystallization from AcOEt and hexanes, the desired product isobtained in the form of a white solid (863 mg; 96%).

[0155] Mass spectrum: 450.4 MH+.

[0156] 5.n)7-(2-(((1,1-dimethylethoxy)carbonyl)amino)-1-oxo-3-((triphenylmethyl)thio)propyl)-2-(2-methoxyphenyl)-8-(phenoxyethyl)-4, 5,6,7-tetrahydro-imidazo-[1.2a]-piperazine

[0157] Intermediate 5.m (850 mg; 1.89 mmol) is treated with a mixture ofTFA (10 ml) containing iPr₃SiH (387 μl, 1.89 mmol) at ambienttemperature for 20 min. The solvents are eliminated under reducedpressure and the crude product is divided between AcOEt (15 ml) and asaturated solution of NaHCO₃ (15 ml). The AcOEt phase is dried overNa₂SO₄, filtered and concentrated under reduced pressure. Thedeprotected product is coupled to Boc-(L)-Cys(Trt)-OH according to amethod similar to that of Stage 1.f (1.26 g; 84%).

[0158] 5.o)7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenoxyethyl)-4,5,6,7-tetrahydro-imidazo-[1,2a]-piperazine

[0159] Product 5 is prepared starting from intermediate 5.n according toa method similar to that of Stage 1.g.

[0160] Mass spectrum: 274.3 MH+. NMR ¹H (DMSO-d6 at 90° C.): 8.5-9.2(3H, s, broad); 7.95-8.1 (1H, d); 7.85-8.0 (1H, s); 7.35-7.5 (1H, m);7.15-7.35 (3H, m); 7.0-7.15 (1H, t); 6.85-7.0 (3H, m); 5.9-6.1 (1H, s,broad); 4.5-4.8 (2H, m, broad); 4.15-4.45 (3H, m, broad); 3.9-4.0 (3H,s); 3.75-4.0 (1H, m, broad); 2.8-3.05 (2H, m, broad); 2.55-2.75 (2H, m,broad).

Example 67-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenoxyethyl)-5,6,7,8-tetrahydro-imidazol[1,2a]pyrazine,dimer: 6:

[0161] Compound 5.o (467 mg; 0.687 mmol) is solubilized in H₂O (25 ml)and the pH of the solution is adjusted to 7.2 by adding a dilute aqueoussolution of NH₄OH. Acetonitrile is added in order to produce a limpidsolution and the mixture is agitated at ambient temperature overnight.The crude product is purified by preparative HPLC on a C18 column usinga gradient of 15 to 40% CH₃CN in TFA at 0.1% over a period of 50minutes. The pure fractions of product are collected and lyophilized.The initial product is lyophilized twice from a dilute solution of HClin order to produce the product in the form of its hydrochloride (161mg; 45%).

[0162] Mass spectrum: 903.5 MH+. NMR 1H (DMSO-d6 at 90° C.): 8.7-9.3(3H, broad s); 7.95-8.1 (1H, d); 7.85-8.0 (1H, s); 7.3-7.5 (1H, t);7.1-7.3 (3H, m); 7.0-7.15 (1H, t); 6.8-7.0 (3H, m); 5.85-6.1 (1H, broads); 4.7-4.9 (1H, broad s); 4.45-4.7 (1H, broad m); 4.1-4.5 (4H, broadm); 3.85-4.0 (4H, s); 3.3-3.5 (2H, broad m); 2.5-2.8 (2H, broad m).

Example 77-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenylsulphonylethyl)-5,6,7,8-tetrahydro-imidazo[1.2a]pyrazine: 7:

[0163] Compound 7 was prepared according to the synthesis diagram below:

[0164] 7.p)7-((1,1-dimethylethoxy)carbonyl)-2-(2-methoxyphenyl)-8-(phenylthioethyl)-5,6,7,8-tetrahydro-imidazo[1,2-a]pyrazine

[0165] Intermediate 5.1 (1.23 g; 3.30 mmol), tri-n-butylphosphine (1.64ml; 6.60 mmol) and phenyldisulphide (1.44 g; 6.60 mmol) are mixed in THF(10 ml). The mixture is agitated at ambient temperature under argon for4 hours. The solvents are eliminated under reduced pressure and thecrude product is purified by flash chromatography on silica gel with anAcOEt:hexanes mixture 1:1 as eluant. The fractions containing theproduct are combined and concentrated under vacuum in order to producethe product in the form of a white foam (1.43 g; 93%).

[0166] Mass spectrum: 466.3 MH+.

[0167] 7.q)7-((1,1-dimethylethoxy)carbonyl)-2-(2-methoxyphenyl)-8-(phenylsulphonylethyl)-5,6,7,8-tetrahydro-imidazo[1,2-a]pyrazine

[0168] Intermediate 7.p (650 mg; 1.40 mmol) is dissolved in CH₂Cl₂ (10ml) and 3-chloroperoxybenzoic acid (483 mg; 2.80 mmol) is added inseveral portions over a period of 10 minutes. The mixture is poured ontoa silica column and eluted with a hexanes:AcOEt mixture 7:3, then ahexanes:AcOEt mixture 1:1 in order to produce the pure product (220 mg;32%).

[0169] Mass spectrum: 498.3 MH+. NMR ¹H (DMSO-d6 at 30° C.): 7.9-8.0(3H, m); 7.7-7.85 (1H, m); 7.6-7.75 (2H, m); 7.45-7.55 (1H,s); 7.15-7.25(1H, m); 6.9-7.1 (2H, m); 5.1-5.25 (1H, t); 4.1-4.3 (1H, broad d);4.0-4.15 (1H, m); 3.8-4.0 (1H, m); 3.85-3.95 (3H, s); 3.6-3.8 (1H, m);3.4-3.6 (1H, m); 3.2-3.4 (H₂O plus a blurred signal); 1.9-2.3 (2H, m);1.3-1.5 (9H, s).

[0170] Stage 7.n)

[0171] Stage 7.n is carried out according to a method similar to Stage5.n. The crude product is used without further purification in thefollowing stage.

[0172] Stage 7.o)

[0173] The stage 7.o is carried out according to a method similar toStage 5.o.

[0174] Mass spectrum: 501.3 MH+.

[0175] Preparation of the Compound of Formula (VII):

[0176] This compound, which is close to those described in PatentApplication PCT WO 97/30053, can be prepared according to the followingsynthesis diagram:

[0177] Pharmacological Part

[0178] In order to illustrate the usefulness of the invention, therefollows a study on the effect of the treatment of a human MCF-7 cellline with the following compounds:

[0179]bis-1,1′-[7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazinedisulphide, designated in this part as compound (I);

[0180]bis-1,1′-7-(2-amino-1-oxo-3-thiopropyl-(2-(1-naphthyl)-8-(2-methylpropyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazin-7-yl),designated in this part as compound (II);

[0181]7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methylphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine,designated in this part as compound (III);

[0182] the compound of formula:

[0183]  designated in this part as compound (IV);

[0184]7-(2-amino-1-oxo-3-thiopropyl)-8-butyl-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine,designated in this part as compound (V);

[0185]bis-1,1′-[7-(2-amino-1-oxo-3-thiopropyl)-2-(methoxyphenyl)-8-(1-methylpropyl)—5,6,7,8-tetrahydroimidazo[1.2a]pyrazine],designated in this part as compound (VI);

[0186] the compound of formula:

[0187]  designated in this part as compound (VII);

[0188] Procedures

[0189] Cell Line

[0190] The MCF-7 cell lines (human pleural cells, breast cancer) wereacquired from the American Tissue Culture Collection (Rockville, Md.,USA).

[0191] Measurement of the Intracellular Quantity of Cyclic AMP for theMCF-7 Cells

[0192] MCF-7 cells (2.10⁴ cells/well) seeded in 24-well plates arecultured for 5 days in Dulbecco's modified Eagle medium (Gibco-Brl,Cergy-Pontoise, France) completed with 10% of foetal calf seruminactivated by heating (Gibco-Brl, Cergy-Pontoise, France), 50000units/I of penicillin and 50 mg/l streptomycin (Gibco-Brl,Cergy-Pontoise, France), and 2 mM of glutamin (Gibco-Brl,Cergy-Pontoise, France). The culture medium is replaced after two washeswith a medium without a serum completed or uncompleted with thespecified agents for a time indicated in the different figures. Agentsactivating the production of cyclic AMP are then added at 37° C. Thereaction is stopped after 30 minutes by suppressing the medium andrapidly adding 100 μl of a 0.1N solution of HCl. These extracts arefrozen at −80° C. until they are used. The concentration of cAMP ismeasured using a commercial measurement kit (reference NEK033 from NEN,Les Ulis, France), following the manufacturer's instructions. Theradioactivity is determined by a Gamma counter (Gamma Master-1277, LKB,Turku, Finland).

[0193] Measurement of in vitro Cell Proliferation

[0194] The MCF-7 cells (3000 cells/well) are cultured in 96-well platesin 80 μl of Dulbecco's modified Eagle medium (Gibco-Brl, Cergy-Pontoise,France) completed with 10% of foetal calf serum inactivated by heating(Gibco-Brl, Cergy-Pontoise, France), 50000 units/I of penicillin and 50mg/l streptomycin (Gibco-Brl, Cergy-Pontoise, France), and 2 mM ofglutamin (Gibco-Brl, Cergy-Pontoise, France) were seeded on a 96-wellplate on day 0. The cells were treated on day 1 for 96 hours withincreasing concentrations of up to 50 μM of each of the compounds to betested. After this period, quantification of cell proliferation isdetermined by a colorimetric test, based on the cleavage of the WST1tetrazolum salt by the mitochondrial dehydrogenases in the viable cells,leading to the formation of formazan (Boehringer Mannheim, Meylan,France). These tests are carried out in duplicate with 8 determinationsper concentration tested. For each compound to be tested, the valuesincluded in the linear part of the sigmoid were retained for linearregression analysis and used to estimate the inhibitory concentration(IC₅₀).

[0195] Measurement of the MAP Kinase Activity

[0196] MCF7 cells (5.105 cells/well) are cultured in 6 wells inDulbecco's modified Eagle medium (Gibco-Brl, Cergy-Pontoise, France)completed with 10% of foetal calf serum inactivated by heat (Gibco-Brl,Cergy-Pontoise, France), a mixture of antibiotics: 50000 units/l ofpenicillin and 50 mg/l of streptomycin (Gibco-Brl, Cergy-Pontoise,France) and 2 mM of glutamin (Gibco-Brl, Cergy-Pontoise, France). After24 hours of culture, the cells are incubated for 48 hours in mediumcontaining no serum in order to return the cells to a state of rest. Thecells are then treated for 1 hour either with compound I or with PD98059(Calbiochem, France Biochem, Meudon, France), a specific inhibitor ofMAP kinase activation. The cells are then stimulated (or not) for 5minutes with 12.5 ng/ml of epidermal growth factor (EGF). The reactionis stopped by two washes with PBS (Gibco-Brl, Cergy-Pontoise, France),at 4° C. containing neither calcium nor magnesium and by adding 150 μlof lysis buffer at 4° C. the composition of which is the following: 10mM of tris, 150 mM of NaCl, 2 mM of EGTA, 2 mM of dithiothreitol, 1 mMof PMSF, 2 mM of orthovanadate, 110 g/ml of leupeptin and 10 μg/ml ofaprotinin. Measurement of the proteins contained in the extracts iscarried out by Bradford's method (Biorad reagents, Ivry-Sur-Seine,France). These extracts are frozen at −80° C. until they are used. Theactivity of the MAP kinase is measured using a commercial measurementkit (reference RPN 84, Amersham Life Science, Les Ulis, France)following the manufacturer's instructions. The radioactivity isdetermined using a Packard scintillation counter (Tricarb 5000CA).

[0197] Equipment

[0198] The vasoactive intestinal peptide (VIP) was acquired from Bachem(Voisins le Bretonneux, France). The choleric toxin, forskolin,isoproterenol, prostaglandin E2 and PD 98059 were acquired fromCalbiochem (France Biochem, Meudon, France). The compounds of formulae(I), (II), (III), (IV), (V), (VI) and (VII) were supplied by BiomeasureInc. (Milford, Mass., USA). All these compounds were used followingtheir manufacturers' recommendations.

[0199] Results

[0200]FIG. 1 shows that activation of the adenylate cyclase by thecholeric toxin (200 ng/ml) or by forskolin (10 μM) leads to a verysignificant increase in the cyclic AMP level. Pretreatment of the cellsfor 30 minutes with 30 μM of comound (I) does not modify the productionof cyclic AMP induced by the direct activator of the adenylate cyclase,forskolin. On the other hand, the production of cyclic AMP stimulated bythe direct activator of the sub-unit, the choleric toxin, is greatlyinhibited by compound (I). This shows that the adenylate cyclase itselfis not modified by compound (I) and that the latter prevents theformation of the heterotrimeric complex.

[0201] VIP has been presented as an extra-cellular ligand of a receptorcoupled with the G protein which stimulates the synthesis of cyclic AMPin human breast cancer cells. FIG. 2 shows that treatment with VIP ofMCF-7 human breast cancer cells increases the intracellular quantity ofcyclic AMP in a concentration-dependent manner. A VIP concentration of10 nM which offers a quasi-optimum production of cyclic AMP is used forthe following tests. This concentration agrees with the data alreadypublished relating to the T47D human breast cancer cell line.

[0202]FIG. 3 shows that a 30 minute pretreatment of the MCF-7 cellsresulting from the in vitro cultures with the compound of formula (I) issufficient to inhibit the accumulation of cyclic AMP cyclic stimulatedby VIP in a concentration-dependent manner. An almost completeinhibition was obtained at a concentration of 100 μM of the compound offormula (I). These results show that a treatment with compound (I) issufficient to block the transduction of the signal the route of whichuses the heterotrimeric G proteins as mediators.

[0203]FIG. 4 shows that a treatment for an hour with the compound offormula (I) is sufficient to modify the response to VIP. Treatments of alonger duration (8 hours and 24 hours) continue to inhibit theproduction of cyclic AMP but the main effect is obtained very rapidly.

[0204] Compound (I) is also capable of inhibiting the formation ofcyclic AMP induced by other agents which stimulate the receptors withseven transmembrane domains. In MCF7 cells, for example, the activity ofthe adenylate cyclase greatly increased by the prostaglandin E₂ isinhibited by a treatment for 30 minutes with compound (I). This suggeststhat treatment of the cells with compound (I) modifies theheterotrimeric form of the G proteins by disassociating the sub-unit ofthe β/γ dimer.

[0205] Inhibition of stimulation by VIP is not restricted to compoundsof a structure analogous to that of the compound of formula (I). Asshown in Table I, compounds (II), (III), (IV), (V), (VI) and (VII)tested in the same model are also capable of reducing the quantity ofcyclic AMP induced by VIP.

[0206] All these results suggest that the compounds tested modulate theactivity of the adenylate cyclase by modifying the heterotrimeric formof the G proteins. Now, it is known that the β/γ dimers can directlyactivate effectors leading to the activation of kinases regulated byextracellular signals (ERK's) or MAP kinases.

[0207]FIG. 6 shows that treatment of the cells for 1 hour with compound(I) doubles the basal activity of the MAP kinase. This suggests that bypreventing the formation of the heterotrimeric complex, compound (I)releases the heterodimer—which itself remains linked to the membrane andactivates the ras route. On the other hand, FIG. 7 shows that afterstimulation of the MAP kinase by the growth factor EGF for 5 minutes,the activity of the enzyme is increased by approximately 7 times.Pretreatment of the cells for 1 hour either with compound (I) or withPD98059, a specific inhibitor of MAP kinase activation, halves theactivity of the MAP kinase. These results suggest that compound (I)stimulates the basal state of the ras route and inhibits this same routeif it is stimulated, thus explainining its anti-proliferative effect.

[0208] Table II in fact shows that compounds (I), (II), (III) and (IV)are capable of inhibiting the in vitro proliferation of MCF7 humantumour cells. TABLE I Compound Inhibition at 30 μM (I) 86% (II) 71%(III) 59% (IV) 52% (V) 68% (VI) 52% (VII) 65%

[0209] Effects of Compounds I, II, III and IV Incubated for 30 Minuteson the Production of Cyclic AMP Stimulated by VIP in MCF7 Cells.

[0210] The cells are incubated for 30 minutes in the presence or not ofcompounds I, II, III and IV (30 μM) which are then stimulated by 10⁻⁸ Mof VIP. The quantification of cyclic AMP is determined byradioimmunoassay. The data represents the average±MSD (n=5 for thecontrol and n=1 for the different compounds). TABLE II Compound testedIC₅₀ (μM) compound I 9.4 compound II 15.0 compound III 16.1 compound IV34.6

[0211] Inhibition of the in vitro Growth of MCF7 Cells by Compounds I,II, III and IV.

[0212] The results of the IC₅₀ are expressed in μM and represent theaverage of 2 experiments.

1. A method of preventing formation of the heterotrimeric G protein in amammal in need thereof comprising administering to said mammal atherapeutically effective dose of a cysteine derivative, characterizedin that said derivative is: either a compound of general formula (A):

 corresponding to sub-formulae (A1) or (A2):

 in which: X represents R₁₂ and Y represents R₈, or X and Y complete aring with 6 members, the X—Y set representing the —CH(R₈)—CH(R₉)—radical; R₁ represents H, a lower alkyl or alkylthio radical; R₂ and R₃represent independently H or a lower alkyl radical; R₄ represents H₂ orO; R₅ represents H, or one of the lower alkyl, lower alkenyl, loweralkynyl, aryl, lower arylalkyl, heterocycle or lower alkyl heterocycleradicals, these radicals being optionally substituted by radicals chosenfrom the group comprising a lower alkyl radical, —O—R₁₀, —S(O)_(m)R₁₀ (mrepresenting 0, 1, or 2), —N(R₁₀)(R₁₁), —N—C(O)—R₁₀, —NH—(SO₂)—R₁₀,—CO₂—R₁₀, C(O)—N(R₁₀)(R₁₁), and —(SO₂)—N(R₁₀)(R₁₁); R₆ and R₇ representindependently H, a —C(O)—NH—CHR₁₃—CO₂R₁₄ radical, or one of the loweralkyl, aryl, lower arylalkyl, heterocycle or lower alkyl heterocycleradicals, these radicals being optionally substituted by radicals chosenfrom the group comprising the OH, alkyl or lower alkoxy, N(R₁₀)(R₁₁),COOH, CON(R₁₀)(R₁₁), and halo radicals, or R₆ and R₇ form together anaryl radical or a heterocycle; R₈ and R₉ represent independently, H, orone of the lower alkyl, aryl, lower arylalkyl, heterocycle or loweralkyl heterocycle radicals, these radicals being optionally substitutedby radicals chosen from the group comprising the OH, alkyl or loweralkoxy, N(R₁₀)(R₁₁), COOH, CON(R₁₀)(R₁₁) and halo radicals, or R₈ and R₉together form an aryl radical or a heterocycle; R₁₀ and R₁₁ representindependently H, an aryl radical or a heterocycle, or an alkyl,arylalkyl or lower alkyl heterocycle radical; R₁₂ represents NR₉, S, orO; R₁₃ represents a lower alkyl radical optionally substituted by aradical chosen from the lower alkyl, —OR₁₀, —S(O)_(m)R₁₀ (m representing0, 1, or 2) and —N(R₁₀)(R₁₁) radicals; R₁₄ represents H or a lower alkylradical; it being understood that the compounds of general formula (A)can also be presented in the form of dimers, when two R₁ radicals eachrepresenting a hydrogen atom are eliminated by oxidization; or acompound of general formula (B) W₁—Ar—W₂  (B)  in which: W₁ represents aremainder originating from a cysteine in reduced or non reduced form; Arrepresents a radical derived from an aminobenzoic acid, the aromaticring of which is optionally substituted; W₂ represents an amino acid,preferably an aliphatic amino acid; or a compound of general formula(C):

 in which: Z₁ represents a lower alkyl radical; Z₂ and Z₃ both representH or Z₂ and Z₃ together form a chain having 2 to 4 elements chosen fromthe —C(O)—, —CH₂—, —CH(NH₂)— and —S— radicals, it being understood thattwo successive elements are not both —C(O)—; it being understood thatthe compounds of general formula (C) can also be presented in the formof dimers, when the radical Z₂ represents an atom of hydrogen which canbe eliminated by oxidization; or also a pharmaceutically acceptable saltof a compound of general formula (A), (B) or (C).
 2. A method oftreating pathologies which result from the formation of theheterotrimeric G protein, except however cancer and proliferativediseases, in a mammal, comprising administering to said mammal atherapeutically effective dose of a cysteine derivative of claim
 1. 3. Amethod according to claim 1, characterized in that the compoundadministered is: either a compound of general formula (A) as defined inclaim 1; or a compound of general formula (B) for which Ar represents aradical derived from an aminobenzoic acid, the aromatic ring of which issubstituted by a phenyl radical and W₁ represents an aliphatic aminoacid;
 4. A method according to claim 1, characterized in that thecompound administered is chosen from one of the following compounds:7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methylphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;7-(2-amino-1-oxo-3-thiopropyl)-8-butyl-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;7-(2-amino-1-oxo-3-thiopropyl)-8-(1-methylpropyl)-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;1-[2(R)-amino-3-mercaptopropyl]-2(S)-n-butyl-4-(1-naphthoyl)piperazine;bis-1,1′-[7-(2-amino-1-oxo-3-thiopropyl)-2-(methoxyphenyl)-8-(1-methylpropyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine]disulphide;bis-1,1′-[7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazinedisulphide;bis-1,1′-7-(2-amino-1-oxo-3-thiopropyl-(2-(1-naphthyl)-8-(2-methylpropyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazin-7-yl)disulphide; the compound of formula:

the compound of formula:

7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-phenyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenylmethoxy)methyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(1-phenylmethoxy)ethyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenoxyethyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methyoxyphenyl)-8-(phenoxyethyl)-5,6,7,8-tetrahydro-imidazo[1.2a]pyrazine,or its dimeric form; and7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenylsulphonylethyl)-5,6,7,8-tetrahydro-imidazo[1.2a]pyrazine;or a pharmaceutically acceptable salt of one of the latter.
 5. A methodaccording to claim 4, characterized in that the compound administered ischosen from one of the following compounds:bis-1,1′-[7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazinedisulphide (I);bis-1,1′-7-(2-amino-1-oxo-3-thiopropyl-(2-(1-naphtyl)-8-(2-methylpropyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazin-7-yl) disulphide (II);7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methylphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine(III); the compound of formula:

7-(2-amino-1-oxo-3-thiopropyl)-8-butyl-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine(V);bis-1,1′-[7-(2-amino-1-oxo-3-thiopropyl)-2-(methoxyphenyl)-8-(1-methylpropyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine]disulphide (VI); the compound of formula:

7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-phenyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;7-(2-amino-1-oxo-3-thiopropyl)-8-(1-methylpropyl)-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;1-[2(R)-amino-3-mercaptopropyl]-2(S)-n-butyl-4-(1-naphthoyl)piperazine;or a pharmaceutically acceptable salt of one of the latter.
 6. A methodaccording to claim 5, characterized in that the compound administered ischosen from one of the following compounds:bis-1,1′-[7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazinedisulphide (I);bis-1,1′-7-(2-amino-1-oxo-3-thiopropyl-(2-(1-naphtyl)-8-(2-methylpropyl)-5,6,7,8-tetrahydroimidazo[1,2a]pyrazin-7-yl)disulphide (II); or a pharmaceutically acceptable salt of one of thelatter.
 7. A method according to claim 2, characterized in that thepathology to be treated is chosen from the pathologies linked to thefollowing biological functions or disorders: smell, taste, perception oflight, neurotransmission, neurodegeneration, endocrine and exocrinegland functions, autocrine and paracrine regulation, arterial tension,viral infection, immunological functions, diabetes and obesity.
 8. Amethod according to claim 2, characterized in that the pathology to betreated is chosen from the following pathologies: cholera, AcquiredImmune Deficiency Syndrome (AIDS), travel diarrhea and familialmasculine precocious puberty.
 9. A compound chosen from the groupconsisting of:7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methylphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-phenyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenylmethoxy)methyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(1-phenylmethoxy)ethyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenoxyethyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methyoxyphenyl)-8-(phenoxyethyl)-5,6,7,8-tetrahydro-imidazo[1.2a]pyrazine,or its dimeric form; and7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenylsulphonylethyl)-5,6,7,8-tetrahydro-imidazo[1.2a]pyrazine.10. Pharmaceutical composition comprising, as an active ingredient, acompound according to claim 9 or a pharmaceutically acceptable salt ofthis compound.
 11. A method of treating pathologies which result fromthe formation of the heterotrimeric G protein in a mammal comprisingadministering to said mammal a therapeutically efficient dose of acompound according to claim
 9. 12. A method according to claim 11,characterized in that the pathology is linked to one of the followingbiological functions or disorders: smell, taste, perception of thelight, neurotransmission, neurodegeneration, endocrine and exocrinegland functions, autocrine and paracrine regulation, arterial tension,embryogenesis, benign cell proliferation, oncogenesis, viral infection,immunological functions, diabetes, obesity, and benign and malignproliferative diseases.